One of the most common causes of cancer is accumulation of double-stranded breaks (DSBs), which occurs frequently during DNA replication. When DNA replication is blocked, replicative helicase activity becomes uncoupled from the newly synthesized daughter strand, resulting in long stretches of single-stranded DNA. The ATR-CHK1 checkpoint pathway recognizes these structures and stabilizes these stalled forks and inhibits cell cycle progression. Loss of ATR allows stalled replication forks to be processed into reversed forks and DSBs, which prevents the re-initiation of replication. It is unclear how stalled replication forks become DSBs in the absence of ATR. Our recent data suggests that PLK1 and RNF4, a SUMO-targeted ubiquitin E3 ligase, drive replisome degradation at sites of replication fork stalling. I hypothesize that PLK1- and RNF4-dependent ubiquitination and removal of replisome components is a naturally occurring process at the G2-M transition which happens prematurely at stalled forks when ATR is absent. Furthermore, I propose that this removal stimulates the recruitment of the SLX4- endonuclease complex, which directly mediates DSB formation at sites of replication fork stalling. To test my models, I will quantify levels of replisome components on chromatin at different phases of the cell cycle and during replication fork stalling in the absence of ATR. In addition, I will develop novel systems t test whether the removal of replisome components is required for fork processing and DSB formation. These experiments will expand our understanding of replication fork collapse and the maintenance of genome integrity. In so doing, our knowledge of cancer etiology and treatment will be extended.